Intronic mirna

Abstract

The present invention relates to miRNA expression constructs, which are useful e.g. in gene and gene-modified cell therapies. In particular, the present invention relates to multiplexed miRNA expression constructs with improved efficiency, wherein multiple miRNA hairpins are expressed from within an intron.

Description

[0001] Intronic miRNA

[0002] Field of the invention

[0003] The present invention relates to miRNA expression constructs, which are useful e.g. in gene and gene-modified cell therapies. In particular, the present invention relates to multiplexed miRNA expression constructs with improved efficiency, wherein multiple miRNA hairpins are expressed from within an intron.

[0004] Background

[0005] Gene and gene-modified cell therapies hold great promise for the treatment and cure of numerous diseases. In many respects, it is the only modality to offer a curative solution, whereas other therapeutic regimens including small molecules and targeted biologies, for example, provide only palliative care. The genetic basis of these therapies relies on either the use of gene augmentation or genomic editing approaches. In the former, a corrected or functional gene is introduced to target cells - typically using a viral vector - with the intent to either replace or augment cellular functionality. In the latter, target cells are either modified to knock out or correct gene function using novel technologies such as gene, base and prime editing.

[0006] Although promising, gene and gene-modified cell therapies still face significant challenges with respect to patient safety and treatment efficacy. The challenges are diverse, relating broadly to (i) transgene expression, (ii) delivery methods, or (iii) genomic editing approaches. For transgene expression, considerations include the choice of gene promoter, DNA sequence optimization, target cell type, and the desired level of expression. For delivery methods, the choice of viral or non-viral vectors should be carefully considered, taking into account payload capacity, target tissues, and risks associated with genotoxicity and immunogenicity. For genomic editing approaches, selecting a fit-for-purpose technology with respect to efficiency and genotoxicity risks is important to measure up forthe intended indication (Fiumara et al., 2024).

[0007] As the industry evolves, there is a growing interest in multiplex engineering capabilities, and specifically in the ability to both express proteins while also silencing (or knocking out) the expression of others. This is typically engineered in a two-step process, where protein expression is achieved through traditional gene augmentation methods using a viral vector, and then separately knocking out other genes using an established gene editing approach (e.g., CRISPR / Cas9, TALE or zinc finger nucleases). This rather complicated two-step approach results in a significant loss in engineering efficiencies and added safety concerns especially with respect to genotoxicity. More recently, gene editing approaches for site specific insertion (SSI) of a donor DNA template (transgene) have also been exploited to circumvent the need for viral vector mediated gene delivery, but this still carries genotoxicity risk and compromised efficiencies when it comes to multiplex engineering needs. Optimization of multiplexed miRNA gene constructs

[0008] We have developed and reported a novel microRNA (miRNA) architecture and gene construct that is capable of highly efficient and tunable gene silencing of one or more targets (Myburgh et al., 2014, 2015; Rousset et al., 2019). We further used this miRNA architecture to create a bimodal (bicistronic) gene construct for co-expression of up to four different proteins. Therefore, through a single gene augmentation step, it uniquely enables the option to simultaneously silence and express multiple genes, without compromising the safety and efficiencies encountered with gene editing approaches (Golinelli et al., 2023; Sakic et al., 2023).

[0009] For a gene augmentation strategy, therapeutic success often relies on the ability to express the transgene(s) to adequate levels. Current gene delivery approaches are very efficient for transfer of small genetic payloads of <2.5 kilobases (kb); however, there is a significant drop in efficiency when larger gene constructs are to be transferred. In principle, increasing genetic payloads not only decreases product / vector manufacturing outputs, but it also substantially compromises gene transfer efficiencies.

[0010] In the original developments of our miRNA gene silencing constructs, the prototype version made use of a ubiquitin promoter and green fluorescent protein (GFP) spacer region (Myburgh et al., 2014). Even though our miRNAs are a mere 107 nucleotides (nt) in length, the entire gene construct was nearly 2,100 nt in size when expressing a single miRNA hairpin. We therefore set out to size-optimize the construct by exchanging the ubiquitin promoter for a truncated version of the elongation factor 1-alpha (EF1 s) and optimizing the spacer region to be both biologically inert and half the length of its predecessor (Rousset et al., 2019). The final result was a construct of 725 nt in length, 65% reduced from its protype. Importantly, miRNA activities were not compromised but were in fact improved against individual targets and furthermore enabled the multiplex capability to silence up to six targets simultaneously (Sakic et al., 2023).

[0011] Use of intronic miRNA for size optimization of gene constructs

[0012] To further size optimize our bimodal gene constructs, we set out to develop a novel gene architecture that would enable the co-expression of multiplexed miRNAs and proteins from a single promoter, rather than in the current architecture utilizing two separate promoters. Notably, it is not possible to co-express miRNAs within or directly upstream or downstream of a protein-coding region, given that Drosha processing of the miRNA destabilizes mRNA transcripts and thus reduces protein expression. We and others have demonstrated this principle with GFP co-expression, where more efficient miRNA processing correlated directly with reduced expression of live GFP (Myburgh et al., 2014; Sun et al., 2006).

[0013] To overcome this challenge, we set out to express multiple miRNAs from within an intron. In nature, most miRNAs are in fact expressed as intronic miRNAs and are thus regulated by the activity of their protein-coding host gene (Y. K. Kim & Kim, 2007; Rodriguez et al., 2004). A well-described example is the miR-106b cluster of three miRNAs expressing from within an intron of the MCM7 gene (Poliseno et al., 2010). Notably, intronic miRNAs are not to be confused with mirtrons, which are short introns that are spliced and debranched into pre-miRNA mimics (Berezikov et al., 2007; Ruby et al., 2007). Mirtrons therefore do not follow canonical miRNA processing by the Drosha enzyme.

[0014] Exploiting the EF1 alpha promoter to express intronic miRNAs

[0015] The human EF1-alpha promoter is one of the most used promoters in expression vectors and human gene therapy applications, owing to its ability to maintain high and stable transgene expression. Since its isolation and characterization in 1989 by Uetsuki and colleagues, it has been subject to substantial modification, resulting in varied activity across many different cell types (D. W. Kim et al., 1990; Milone et al., 2009; Uetsuki et al., 1989; Zheng & Baum, 2014). A notable feature of the EF1-alpha gene is the presence of a non-coding first exon, with the transcription initiation site being within the second exon of the gene. Moreover, the first intron of EF1-alpha carries additional activation sites that drives higher expression of a gene of interest (D. W. Kim et al., 1990; Wakabayashi-lto & Nagata, 1994). When the gene of interest is fused into the first exon, appreciable promoter activity is still reported, although not to the levels observed for the fully constituted EF1-alpha promoter. The truncated version of the EF1-alpha promoter (excluding the first intron) is referred to as EF1 short, or EFS.

[0016] Given our goal to express intronic miRNAs, we explored the option of exploiting the unique features and properties of the EF1-alpha promoter. More specifically, with the first intron being retained, we created multiple novel versions of the promoter wherein we adapted the intronic sequence to express our optimized miRNA architecture (architecture described in Myburgh et al., 2014).

[0017] Summary of the invention

[0018] In an aspect, the present invention provides a microRNA (miRNA) expression construct comprising a promoter operably linked to an expressed transcript, wherein the promoter comprises a promoter element and an intronic element, wherein the intronic element comprises one or more miRNA hairpins.

[0019] In some embodiments, the intronic element is an elongation factor 1-alpha (EF1 a) intron, derivative or fragment thereof.

[0020] In some embodiments, the intronic element comprises the sequence set forth in SEQ ID NO: 3 or a sequence having at least 80% identity thereto.

[0021] In some embodiments, the promoter is an EF1 a promoter, a derivative of a EF1 a promoter or a EF1 short promoter,

[0022] In some embodiments, the promoter comprises the sequence set forth in SEQ ID NO: 1 or a sequence having at least 80% identity thereto.

[0023] In some embodiments, the intronic sequence comprises at least four, or at least six miRNA hairpins In some embodiments, the miRNA hairpins target at least two, at least three, at least four, at least five or at least six different transcripts or genes.

[0024] In some embodiments, each of the miRNA hairpins functionally modulates a target gene, optionally wherein each of the miRNA hairpins provides a level of functional modulation of its target gene that is the same or equivalent to the miRNA expression construct that only comprises that miRNA hairpin.

[0025] In an aspect, the present invention provides a nucleic acid comprising the miRNA expression construct of the invention.

[0026] In an aspect, the present invention provides a plasmid comprisingthe miRNA expression construct or nucleic acid of the invention.

[0027] In an aspect, the present invention provides a vector comprising the miRNA expression construct, nucleic acid or plasmid of the invention.

[0028] In an aspect, the present invention provides a host cell comprising the miRNA expression construct, nucleic acid, plasmid orvector of the invention.

[0029] In an aspect, the present invention provides a composition comprising the miRNA expression construct, nucleic acid, plasmid, vector or host cell of the invention.

[0030] In an aspect, the present invention provides a method for reducing expression of a gene in a cell comprising expressing the miRNA expression construct, nucleic acid, plasmid or vector of the invention in the cell.

[0031] In an aspect, the present invention provides a method for preparing an engineered cell, optionally an immune effector cell, from a patient or healthy donor comprising:

[0032] (a) collecting a cell, e.g. from the patient;

[0033] (b) gene modifying the cell to generate an engineered cell: i) with a chimeric antigen receptor or a T cell receptor to; and simultaneously ii) with the multiplexed miRNA from the same expression construct, nucleic acid, plasmid orvector of the invention.

[0034] In an aspect, the present invention provides an engineered cell or an engineered effector cell obtainable by the method of the invention.

[0035] In an aspect, the present invention provides the miRNA expression construct, nucleic acid, plasmid, vector, composition or host cell of the invention, for use in therapy.

[0036] In an aspect, the present invention provides a method of treating cancer, an infectious disease, an auto-immune disease or an inherited disorder, comprising administering the miRNA expression construct, nucleic acid, plasmid, vector, composition, or host cell of the invention. In an aspect, the present invention provides a method of making a miRNA expression construct, comprising: a) providing an intronic element sequence; b) modifying the intronic element sequence by inserting one or more miRNA hairpins into the intronic element sequence; c) incorporating the intronic element sequence and a promoter element into a promoter; and d) providing a nucleic acid comprising the promoter operably linked to an expressed transcript.

[0037] Brief description of the drawings

[0038] Figure 1. Schematic representation of anti-CD7 CAR gene constructs co-expressing intronic miRNA. A. Gene construct from lentivector plasmid pATN1376, containing a modified version of the human EF1-alpha 1 promoter which carries intronic miRNAs targeting the CD3^ subunit of the T-cell receptor (TCR), 2M and CD7. The modified promoter drives expression of a second generation anti-CD7 CAR and a reporter gene (RQR8 in this case), separated by a 2A peptide. The CD7 CAR includes a CD3 cytoplasmic signal sequence, an intracellular costimulatory region derived from 4-1 BB (CD137), a CD8 transmembrane region and a VHH-based CD7-targeting domain. B. Gene construct from lentivector plasmid pATN1377, which is identical to pATN1376, except that is includes two additional miRNAs targeting CD5 and OITA. CAR, Chimeric antigen receptor; LS, Leader / signal peptide sequence; TM, Transmembrane domain; UTR, Untranslated region; VHH, Heavy chain only binder.

[0039] Figure 2. Expression of CD7 CAR and silencing by intronic miRNAs in primary T-cells. A. Representative flow cytometry dot plots of CD7 CAR T-cells expressing four intronic miRNAs (1376), following the depletion of TCRa / p positive T-cells. The percentage positive CD7 CAR T-cells and corresponding silencing of the TCR, HLA-I and CD7 is shown in B and C (n=3 donors). CAR positive cells were detected based on direct CAR detection with recombinant CD7 protein or RQR8 co-expression (CD34). Remaining cells after TCRa / p depletion are devoid of TCRa / p, silenced for HLA-I and CD7, and fully express RQR8 reporter gene (CD34), as well as the CAR7 protein. D. Representative flow cytometry dot plots of CD7 CAR T-cells expressing six intronic miRNAs (1377), following depletion of TCRa / p positive T-cells. The percentage positive CD7 CAR T-cells and corresponding silencing of the TCR, HLA-I, CD7, HLA-II and CD5 is shown in E and F (n=3 donors). CAR positive cells were detected based on direct CAR detection with recombinant CD7 protein or RQR8 co-expression (CD34). Remaining cells afterTCRa / p depletion are devoid of TCRa / p, silenced for HLA-ABC, CD7, HLA-II and CD5, and fully express RQR8 reporter gene (CD34), as well as the CAR7 protein.

[0040] Figure 3. Functional activity of CD7 CAR T-cells co-expressing intronic miRNA. A and B. Specific cytotoxicity of MOLT-4 tumor cells. Engineered CD7 CAR T-cells (effector, E) and MOLT-4 cells (target, T) were co-cultured at E:T ratios of 1 :1 and rechallenged with target cells every 3-4 days upon successful elimination of the tumor cells. Target cell survival (MOLT-4 cells labelled with CTV) was assessed by detection of GFP positivity on flow cytometry after the indicated time points. Both CD7 CAR T-cell populations, expressing either 4 x intronic miRNAs (1376) or 6 x intronic miRNAs (1377) were able to eliminate MOLT-4 cells over three repeat stimulations. C and D. Specific cytotoxicity of SUP-T1 tumor cells. Engineered CD7 CAR T-cells (effector, E) and SUP-T1 cells (target, T) were co-cultured at E:T ratios of 1 :1 and re-challenged with target cells every 3-4 days upon successful elimination of the tumor cells. Target cell survival (SUPT1 cells labelled with CTV) was assessed by detection of GFP positivity on flow cytometry after the indicated time points. Both CD7 CAR T-cell populations, expressing either 4 x intronic miRNAs (1376) or 6 x intronic miRNAs (1377) were able to eliminate SUP-T1 cells over two repeat stimulations.

[0041] Figure 4. Schematic representation of anti-CD19 CAR gene constructs coexpressing intronic miRNA. Gene construct from lentivector plasmid pATN856, containing a modified version of the human EF1 -alpha 1 promoter which carries intronic miRNAs targeting CD52, 2M and the CD3z subunit of the T-cell receptor (TCR). The modified promoter drives expression of a second generation anti-CD19 CAR and a reporter gene (RQR8 in this case), separated by a 2A peptide. The CD19 CAR includes a CD3z cytoplasmic signal sequence, an intracellular costimulatory region derived from 4- 1 BB (CD137), a CD8 transmembrane region and a scFv-based CD19-targeting domain. Downstream the modified EF1-alpha promoter, another constitutive promoter, hPGK, is driving a complementary reporter gene mCherry. CAR, Chimeric antigen receptor; LS, Leader / signal peptide sequence; TM, Transmembrane domain; UTR, Untranslated region; scFv, single-chain variable fragment.

[0042] Figure 5. Expression of CD19 CAR and silencing by intronic miRNAs in primary T- cells. A and B. Representative flow cytometry dot plots of CD19 CAR T-cells expressing three intronic miRNAs (856). The percentage positive CD19 CAR T-cells through the reporter gene RQR8, the percentage of positive mCherry T-cells based on PGK promoter activity and the corresponding silencing of the CD52, HLA-I and TCR is shown in C, D and E (n=2 donors). CAR positive cells were detected based on direct detection of RQR8 coexpression (using CD34 antibody). Approximately 59.8% of the cells are RQR8 positive, and 62.5% are mCherry positive (hPGK promoter on second position). CD52 protein expression is reduced by an average of 66.5%, while HLA-I and TCR show silencing efficiencies of 47% and 70%, respectively.

[0043] Figure 6. Functional activity of CD19 CAR T-cells co-expressing intronic miRNA.

[0044] A and B. Specific cytotoxicity of Jeko-GFP tumor cells. Engineered CD19 CAR T-cells (effector, E) and Jeko cells (target, T) were co-cultured at E:T ratios of 3:1 (A), 1 :3 and 1 :1 (B) and tumor cells amount was assessed at 24 and 48 hours (A) or 24 and 72 hours (B). Target cell survival (Jeko-GFP cells) was assessed by detection of GFP positivity on flow cytometry after the indicated time points. A. CD19 CAR T-cell populations, expressing 3 x intronic miRNAs (856) was able to eliminate Jeko-GFP cells after 2 days with a E:T ratio of 3:1 . B. CD19 CAR T-cell populations, expressing 3 x intronic miRNAs (856) were able to eliminate Jeko-GFP cells after 3 days at E:T ratio of 1 :1 . And at an E:T ratio of 1 :3, CD19 CAR T-cell populations, expressing 3 x intronic miRNAs (856), was able to control tumor growth. Detailed description of the invention miRNA expression construct

[0045] In an aspect, the present invention provides a microRNA (miRNA) expression construct comprising a promoter operably linked to an expressed transcript, wherein the promoter comprises a promoter element and an intronic element, wherein the intronic element comprises one or more miRNA hairpins.

[0046] In embodiments, it will generally be understood that the miRNA hairpins and expressed transcript are both operably linked by the promoter. By “operably linked”, it is meant that the expression of the miRNA hairpins (transcription) and the expressed transcript (transcription and ultimately translation) are under the control of the promoter.

[0047] In embodiments, it will generally be understood that the “promoter element” comprises the minimum sequences necessary for the function of the promoter, i.e. to initiate transcription, cause expression and express the miRNA hairpins and expressed transcript. In other terms, the “promoter element” is a core promoter.

[0048] In embodiments, it will generally be understood that an “intronic element” is an intron, fragment or derivative thereof. Any known intron or derivative may be used. In a preferred embodiment, the intronic element is an intron. In embodiments, it will generally be understood that the miRNA hairpin sequences are inserted into the sequence of the intronic element.

[0049] In most general embodiments, “miRNA hairpins” encompass any sequence capable of causing RNA interference, e.g. miRNA, siRNA or shRNA. In some embodiments, “miRNA hairpins” are understood as the functional unit of miRNA, capable of causing modulation of a target gene or transcript via RNA interference. In some such embodiments, miRNA hairpins generally comprise a stem-loop structure comprising the target sequence, complementary sequence and additional sequences associated with miRNA processing. In some embodiments, miRNA hairpins are also referred to as inhibitory nucleic acids herein. In some embodiments, miRNA are not siRNA or shRNA.

[0050] In some embodiments, the miRNA expression construct is a nucleic acid, such as a nucleic acid comprising DNA, RNA or both DNA and RNA. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA.

[0051] In some embodiments, the intronic element is an elongation factor 1-alpha (EF1 a) intron, derivative or fragment thereof. In some embodiments, the intronic element is a human EF1 a intron (e.g. from GenBank J04617.1 ), derivative or fragment thereof. In some embodiments, the intronic element is a first intron of, e.g. human, EF1 a, or a derivative or fragment thereof.

[0052] In some embodiments, the intronic element comprises the sequence set forth in SEQ ID NO: 3 or a sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto. In some embodiments, the promoter is an EF1 a promoter, a derivative of a EF1 a promoter or a EF1 short promoter, optionally wherein the EF1 a promoter comprises the sequence set forth in SEQ ID NO: 1 or a sequence having 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto.

[0053] In some embodiments, the promoter is a eukaryotic promoter, such as a Pol II or Pol III promoter, an inducible promoter, tissue-specific promoter, cell lineage-specific promoter, a synthetic promoter, and / or a promoter listed in Table 3 or a UBI promoter.

[0054] In some embodiments, the promoter is a synthetic promoter further comprising an elongation factor 1-alpha (EF1 a) intron, derivative or fragment thereof, optionally comprising SEQ ID NO: 3 or a sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto.

[0055] In some embodiments, the intronic element comprises less than 620 base pairs. In some embodiments, the intronic element comprises 618 base pairs.

[0056] In some embodiments, the promoter comprises the sequence set forth in SEQ ID NO: 1 or a sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto.

[0057] In some embodiments, the promoter element comprises the sequence set forth in SEQ ID NO: 3 or a sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto.

[0058] In embodiments, it will generally be understood that the insertion of the miRNA hairpins is not taken into account for the calculation of the sequence identity of the promoter or intronic element sequence into which the miRNA hairpins have been inserted, i.e. the sequence, and any percentage identity thereto, is determined before the miRNA hairpins are inserted.

[0059] Throughout the present specification various sequences are described, such as the sequences of intronic elements and promoters comprised within the promoter of the expression construct. For the avoidance of doubt, any reference to the sequences of these elements refers to these sequences prior to the insertion of one or more miRNA hairpins in order to form the microRNA expression construct of the invention. For example, the statements that “the intronic element comprises the sequence set forth in SEQ ID NO: 3 or a sequence having at least 80% identity thereto” indicates that the intronic element itself comprises the sequence set forth in SEQ ID NO: 3 or a sequence having at least 80% identity thereto. This sequence will, of course, be altered once the miRNA hairpins have been inserted, i.e. the sequence, and any percentage identity thereto, is determined before the miRNA hairpins are inserted.

[0060] Accordingly, in embodiments, the intronic element comprises the sequence set forth in SEQ ID NO: 3 or a sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto, into which the miRNA hairpins have been inserted. In some embodiments, the promoter comprises the sequence set forth in SEQ ID NO: 1 or a sequence having 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto, into which the miRNA hairpins have been inserted. In some embodiments, it can also be considered that the intronic sequence or promoter respectively further comprises the miRNA hairpin sequences.

[0061] Equally, sequences used for insertin the miRNA hairpins into the intronic element, e.g. restriction enzyme sites and residual nucleotides from restriction enzyme sites, are not taken into account for the calculation of the sequence identity of the promoter or intronic element sequence into which the miRNA hairpins have been inserted.

[0062] In some embodiments, the intronic sequence comprises at least two, three four, five, six, seven, eight, nine, ten or more than ten miRNA hairpins. In some embodiments, the intronic sequence comprises at least four or at least six miRNA hairpins. In some embodiments, the intronic sequence comprises three miRNA hairpins. In some embodiment, the intronic sequence comprises four miRNA hairpins. In some embodiments, the intronic sequence comprises six miRNA hairpins.

[0063] In some embodiments, the miRNA hairpins target at least two, at least three, at least four, at least five or at least six different transcripts or genes.

[0064] In some embodiments, each of the miRNA hairpins functionally modulates a target gene, optionally wherein each of the miRNA hairpins provides a level of functional modulation of its target gene that is the same or equivalent to the miRNA expression construct that only comprises that miRNA hairpin. In embodiments, “the miRNA expression construct that only comprises that miRNA hairpin” is generally understood to be the equivalent, single-miRNA-hairpin expression construct. Thus, it is understood that multiplexed miRNA hairpins in the miRNA expression construct of the invention can retain the same efficacy as equivalent single miRNA hairpin constructs. In embodiments, “functionally modulates” can comprise downregulation or upregulation, e.g. increased expression or decreased expression. In some embodiments, the functional modulation is knockdown or silencing. In some embodiments, the functional modulation is upregulation by knockdown or silencing of a repressor / inhibitory target gene / mRNA.

[0065] In some embodiments, the intronic element comprises miRNA hairpins targeting one or more of CD3z, B2M, CD7, CD5 and / or CIITA. In some embodiments, the intronic element comprises miRNA hairpins targeting one or more of CD3z, CD52, B2M, CD7, CD5 and / or CIITA.

[0066] In some embodiments, the intronic element comprises a first and second miRNA hairpin targeting CD3z, a miRNA hairpin targeting B2M and a miRNA hairpin targeting CD7, optionally wherein the intronic sequence further comprises a miRNA hairpin targeting CD5 and a miRNA hairpin targeting CIITA. In some embodiments, the intronic element comprises a miRNA hairpin targeting CD52, a miRNA hairpin targeting B2M and a miRNA hairpin targeting CD3z.

[0067] In some embodiments, a miRNA hairpin targeting CD3z comprises a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4, or a fragment thereof; and / or a miRNA hairpin targeting CD3z comprises a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5, or a fragment thereof; and / or a hairpin targeting CD52 comprises a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 12, or a fragment thereof; and / or a hairpin targeting B2M comprises a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6, or a fragment thereof; and / or a hairpin targeting CD7 comprises a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7, or a fragment thereof; and / or a hairpin targeting CD5 comprises a sequence having 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8, or a fragment thereof; and / or a hairpin targeting CIITA comprises a sequence having 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 9, or a fragment thereof.

[0068] In some embodiments, the miRNA expression construct comprises at least two different miRNA hairpins which target different regions of the same transcript, and / or which target different transcripts or splice variants of the same gene.

[0069] In some embodiments, the expressed transcript comprises a sequence encoding a moiety for re-directing immune effector cell function.

[0070] In some embodiments, the miRNA expression construct comprises a sequence encoding an engineered T cell receptor, optionally a chimeric antigen receptor.

[0071] In some embodiments, the chimeric antigen receptor specifically binds to CD7, optionally wherein the chimeric antigen receptor comprises an anti-CD7 VHH.

[0072] In some embodiments, the chimeric antigen receptor specifically binds to CD19, optionally wherein the chimeric antigen receptor comprises an anti-CD19 scFv. In some embodiments of this type, the intronic element comprises a miRNA hairpin targeting CD52, a miRNA hairpin targeting B2M and a miRNA hairpin targeting CD3z. In some embodiments of this type, the miRNA expression construct is capable of controlling or controls tumor growth. In some embodiments of this type, the miRNA expression construct is capable of inhibiting or inhibits, e.g. therapeutically inhibits, tumor growth. In some embodiments of this type, the miRNA expression construct is capable of eliminating or eliminates (e.g. kills) tumor cells. In some embodiments, the chimeric antigen receptor is a bispecific chimeric antigen receptor or comprised of tandemly expressed chimeric antigen receptors for dual targeting of antigens on tumor tissues.

[0073] In some embodiments, the construct further comprises a selection gene, optionally LNGFR, truncated endothelial growth factor receptor (tEGFR), tCD19, tCD20, tCD34 or a derivative thereof.

[0074] In some embodiments, the construct further comprises a suicide gene, optionally herpes simplex virus thymidine kinase (HSV-tk), inducible caspase 9 (iCasp9), truncated endothelial growth factor receptor (tEGFR), RQR8, dihydrofolate reductase (DHFR), tCD20 and thymidylate synthase (TYMS).

[0075] In some embodiments, the construct further comprises an internal ribosome entry site (IRES).

[0076] In some embodiments, the construct further comprises a peptide cleavage site, optionally a 2A peptide, optionally 2A, P2A, T2A, E2A, F2A, BmCPV 2A, and BmIFV 2A.

[0077] In some embodiments, the construct achieves knockdown or functional silencing of at least one, two, three, four, five or six genes.

[0078] In a preferred embodiment, the miRNA hairpins target a combination of CD3z, B2M, CD7, CD5 and CIITA genes. In a preferred embodiment, the miRNA hairpins target a combination of CD3z, CD52, B2M, CD7, CD5 and CIITA genes. The miRNA expression constructs can be defined as comprising a first, and second different miRNA hairpin and optionally one or more of a second, third, fourth, fifth and sixth further different miRNA. Unless otherwise specified, references to “first”, “second”, “third”, “fourth”, “fifth” and “sixth” miRNA hairpins are used to refer to the multiple different miRNA hairpins comprised within these constructs, and do not necessarily indicate anything regarding the order or position of the miRNA hairpins within the construct, e.g. a “first” miRNA hairpin is not necessarily located 5’ of a “second” miRNA hairpin. However, in a particular embodiment, the first, second, third, fourth, fifth and sixth miRNA hairpins are present in the stated order, 5’ to 3’.

[0079] In some embodiments, with reference to the miRNA hairpins targeting CD3z, CD52, B2M, CD7, CD5 and CIITA, the sequences of the miRNA hairpins can be further defined as follows:

[0080] Table 1 : miRNA hairpin sequences

[0081] In Table 1 , the first column indicates the target protein / gene in question; the second column indicates the identifier of the target sequence, and the third column indicates the sequence which preferably may be targeted by any miRNA expression construct of the present invention which targets the gene indicated in the first column. Thus, it is understood that, for any miRNA expression construct of the present invention which includes a miRNA hairpin targeting CD3z, the miRNA hairpin targeting CD3z can preferably comprise any of CD3z_T1 or CD3z_T2, which are defined by the sequences provided in the third column.

[0082] In an embodiment, the multiplexed miRNA expression construct of the present invention targeting a gene listed in Table 6 comprises a sequence with 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence listed in Table 6. In an embodiment, the multiplexed miRNA expression construct of the present invention targeting a gene listed in Table 6 comprises a sequence having five, four, three, two, one or zero mismatches to a sequence listed in Table 6. Preferably with the multiplexed miRNA expression construct of the present invention, this can be the case for all of the named genes which are targeted, such as all two, three, four, five or six of the named genes which are targeted.

[0083] In a further embodiment of the present invention with reference to the genes that may be targeted in the present invention, the transcripts of these genes which are targeted, i.e. the target transcripts, can be further defined as in Table 2 below.

[0084] Table 2: Target transcripts

[0085]

[0086] In Table 2, the first column indicates the target protein / gene in question, and the second and third columns indicate the transcript of that gene which is preferably to be targeted by any miRNA expression construct of the present invention which targets the gene indicated in the first column. The second column provides a transcript by reference to the NCBI database, and the third column defines a transcript by reference to the ENSEMBL database. These database references correspond to the published main versions of the databases accessible on 3 May 2022, i.e. Ensembl Release 106. Thus, it is understood for example that, for any miRNA expression construct of the present invention which includes a miRNA hairpin targeting B2M, the miRNA hairpin targeting B2M can preferably target any of the NM_004048, ENST00000648006 and ENST00000559916 transcripts. Thus, the hairpin targeting B2M preferably targets a sequence comprised within one of the listed transcripts. This applies for every gene listed in Table 2 and the associated transcripts.

[0087] In an embodiment, the multiplexed miRNA expression construct of the present invention targeting a gene listed in Table 2 targets a transcript with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a transcript listed in Table 2. The transcript in question is that which corresponds to the gene that is targeted. Preferably with the multiplexed miRNA expression construct of the present invention, this can be the case for all of the named genes which are targeted, such as all two, three, four, five or six of the named genes which are targeted.

[0088] In some embodiments, the miRNA expression construct comprises the sequence set forth in SEQ ID NO: 10 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto.

[0089] In some embodiments, the miRNA expression construct comprises the sequence set forth in SEQ ID NO: 11 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto.

[0090] In some embodiments, the miRNA expression construct comprises the sequence set forth in SEQ ID NO: 12 or a sequence having 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto.

[0091] In some embodiments, the miRNA expression construct comprises the sequence set forth in SEQ ID NO: 13 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto.

[0092] Other forms

[0093] In an aspect, the present invention provides a nucleic acid comprising the miRNA expression construct of the invention. In some embodiments, the nucleic acid comprises or consists of DNA. In some embodiments, the nucleic acid comprises or consists of RNA.

[0094] In an aspect, the present invention provides a plasmid comprisingthe miRNA expression construct or nucleic acid of the invention.

[0095] In an aspect, the present invention provides a vector comprising the miRNA expression construct, nucleic acid or plasmid of the invention.

[0096] In some embodiments, the vector is an expression vector, preferably an adenovirus, an adeno-associated virus, a retrovirus or a lentivirus vector.

[0097] In some embodiments, the vector further comprises at least one drug resistance marker.

[0098] In an aspect, the present invention provides a host cell comprising the miRNA expression construct, nucleic acid, plasmid orvector of the invention.

[0099] In some embodiments, the host cell is a eukaryotic cell, preferably a mammalian cell, preferably an immune effector cell.

[0100] In some embodiments, the immune effector cell is selected from the group comprising: alpha-beta T cells, gamma-delta T-cells, tumour infiltrating lymphocytes (TILS), TCR- engineered T cells, CAR T cells, NK cells, NK / T cells, T regulatory cells, monocytes and macrophages. In some embodiments, the host cell is a T-cell or a CAR T-cell.

[0101] In an aspect, the present invention provides a composition comprising the miRNA expression construct, nucleic acid, plasmid, vector or host cell of the invention.

[0102] In some embodiments, the composition further comprises one or more pharmaceutically acceptable adjuvants, diluents, excipients or salts.

[0103] Applications and methods

[0104] In an aspect, the present invention provides a method for reducing expression of a gene in a cell comprising expressing the miRNA expression construct, nucleic acid, plasmid or vector of the invention in the cell.

[0105] In some embodiments, expressing the miRNA expression construct, nucleic acid, plasmid or vector in the cell comprises transfecting the cell with the miRNA expression construct, nucleic acid, plasmid or vector.

[0106] In some embodiments, expressing the miRNA expression construct, nucleic acid, plasmid or vector in the cell comprises introducing the miRNA expression construct, nucleic acid, plasmid or vector into the cell via electroporation.

[0107] In some embodiments, expressing the miRNA expression construct, nucleic acid, plasmid or vector in the cell comprises transfecting the cell with a transposon comprising the miRNA expression construct, nucleic acid, plasmid or vector, optionally wherein the transposon is sleeping beauty, piggyBAC orTol2.

[0108] In some embodiments, the cell is a eukaryotic cell, preferably a human cell, preferably an immune effector cell.

[0109] In some embodiments, the method is an in vivo, in vitro or ex vivo method.

[0110] In some embodiments, the method comprises transplanting the cell into an organism.

[0111] In some embodiments, the cell is comprised in an organism.

[0112] In an aspect, the present invention provides a method for preparing an engineered cell, optionally an immune effector cell, from a patient or healthy donor comprising:

[0113] (a) collecting a cell, e.g. from the patient;

[0114] (b) gene modifying the cell to generate an engineered cell: i) with a chimeric antigen receptor or a T cell receptor; and ii) with the multiplexed miRNA from the same expression construct, nucleic acid, plasmid or vector of the invention.

[0115] In some embodiments, steps i) and ii) are simultaneous. In an aspect, the present invention provides an engineered effector cell obtainable by the method of the invention.

[0116] In some embodiments, the miRNA expression construct, nucleic acid, plasmid or vector is inserted into the cell by a TALE nuclease, megaTAL, Zinc Finger Nuclease, or CRISPR, optionally CRISPR / Cas9.

[0117] In some embodiments, the miRNA expression construct, nucleic acid, plasmid or vector is expressed in the cell from an expression vector, optionally an adenovirus, an adeno- associated virus, a retrovirus or a lentivirus vector.

[0118] In some embodiments, the miRNA expression construct, nucleic acid, plasmid or vector is expressed in the cell from a transposon, optionally wherein the transposon is sleeping beauty, piggyBAC orTol2.

[0119] In an aspect, the present invention provides the miRNA expression construct, nucleic acid, plasmid, vector, composition or host cell of the invention, for use in therapy.

[0120] In an aspect, the present invention provides the miRNA expression construct, nucleic acid, plasmid, vector, composition, or host cell of the invention, for use in a method of treating cancer, an infectious disease, an auto-immune disease or an inherited disorder.

[0121] In an aspect, the present invention provides a method of treating cancer, an infectious disease, an auto-immune disease or an inherited disorder, comprising administering the miRNA expression construct, nucleic acid, plasmid, vector, composition, or host cell of the invention.

[0122] In an aspect, the present invention provides the miRNA expression construct, nucleic acid, plasmid, vector, composition, or host cell of the invention, for use in a method of manufacturing a medicament.

[0123] In an aspect, the present invention provides the miRNA expression construct, nucleic acid, plasmid, vector, composition, or host cell of the invention, for use in a method of manufacturinga medicamentforthetreatment of cancer, an infectious disease, an autoimmune disease or an inherited disorder.

[0124] In an aspect, the present invention provides a method of making a miRNA expression construct, comprising: a) providing an intronic element sequence; b) modifying the intronic element sequence by inserting one or more miRNA hairpins into the intronic element sequence; c) incorporating the intronic element sequence and a promoter element into a promoter; and d) providing a nucleic acid comprising the promoter operably linked to an expressed transcript. Description of general aspects

[0125] RNA inhibition

[0126] An inhibitory nucleic acid may inhibit the transcription of a gene or prevent the translation of a gene transcript in a cell. An inhibitory nucleic acid may be from 16 to 1000 nucleotides long, and in certain embodiments from 18 to 100 nucleotides long. In certain embodiments, the inhibitory nucleic acid is an isolated nucleic acid that binds or hybridizes to a gene of interest.

[0127] Inhibitory nucleic acids are well known in the art. For example, siRNA, shRNA and doublestranded RNA have been described in U.S. Patents 6,506,559 and 6,573,099, as well as in U.S. Patent Publications 2003 / 0051263, 2003 / 0055020, 2004 / 0265839, 2002 / 0168707, 2003 / 0159161 , and 2004 / 0064842, all of which are herein incorporated by reference in their entirety.

[0128] Since the discovery of RNAi by Fire and colleagues in 1998, the biochemical mechanisms have been rapidly characterized. Double stranded RNA (dsRNA) is cleaved by Dicer, which is an RNAase III family ribonuclease. This process yields miRNAs of ~21 nucleotides in length. These miRNAs are incorporated into a multiprotein RNA-induced silencing complex (RISC) that is guided to target mRNA. RISC cleaves the target mRNA in the middle of the complementary region. In mammalian cells, the related miRNAs are found that are short RNA fragments (~22 nucleotides). miRNAs are generated after Dicer- mediated cleavage of longer (~70 nucleotide) precursors with imperfect hairpin RNA structures. The miRNA is incorporated into a miRNA-protein complex (miRNP), which leads to translational repression of target mRNA.

[0129] In designing RNAi there are several factors that may considered such as the nature of the siRNA, the durability of the silencing effect, and the choice of delivery system. To produce an RNAi effect, the miRNA that is introduced into the organism may typically contain exonic sequences. Furthermore, the RNAi process is homology dependent, so the sequences are often carefully selected so as to maximize gene specificity, while minimizing the possibility of cross-interference between homologous, but not genespecific sequences. Particularly, the miRNA often exhibits greater than 80, 85, 90, 95, 98% or even 100% identity between the sequence of the miRNA and a portion of the nucleotide sequence of a target gene. Sequences less than about 80% identical to the target gene may be substantially less effective. Thus, the greater identity between the miRNA and the target gene to be inhibited, the less likely expression of unrelated genes will be affected.

[0130] In addition, the size of the miRNA is an important consideration. In some embodiments, the present invention relates to miRNA molecules that include at least about 19-25 nucleotides, and are able to modulate target gene expression. In the context of the present invention, the miRNA is particularly less than 500, 200, 100, 50, 25, 24, 23 or 22 nucleotides in length. In some embodiments, the miRNA is from about 25 nucleotides to about 35 nucleotides or from about 19 nucleotides to about 25 nucleotides in length.

[0131] To improve the effectiveness of miRNA-mediated gene silencing, guidelines for selection of target sites on mRNA have been developed for optimal design of miRNA (Soutschek et aL, 2004; Wadhwa et aL, 2004). These strategies may allow for rational approaches for selecting siRNA sequences to achieve maximal gene knockdown. To facilitate the entry of miRNA into cells and tissues, a variety of vectors including plasmids and viral vectors such as adenovirus, lentivirus, and retrovirus have been used (Wadhwa et al., 2004).

[0132] Within an inhibitory nucleic acid, the components of a nucleic acid need not be of the same type or homogenous throughout (e.g., an inhibitory nucleic acid may comprise a nucleotide and a nucleic acid or nucleotide analog). Typically, an inhibitory nucleic acid forms a double-stranded structure; the double-stranded structure may result from two separate nucleic acids that are partially or completely complementary. In certain embodiments of the present invention, the inhibitory nucleic acid may comprise only a single nucleic acid (polynucleotide) or nucleic acid analog and form a double-stranded structure by complementing with itself (e.g., forming a hairpin loop). The doublestranded structure of the inhibitory nucleic acid may comprise 16 - 500 or more contiguous nucleobases, including all ranges therebetween. The inhibitory nucleic acid may comprise 17 to 35 contiguous nucleobases, more particularly 18 to 30 contiguous nucleobases, more particularly 19 to 25 nucleobases, more particularly 20 to 23 contiguous nucleobases, or 20 to 22 contiguous nucleobases, or 21 contiguous nucleobases that hybridize with a complementary nucleic acid (which may be another part of the same nucleic acid or a separate complementary nucleic acid) to form a double-stranded structure. miRNA can be obtained from commercial sources, natural sources, or can be synthesized using any of a number of techniques well-known to those of ordinary skill in the art. For example, commercial sources of predesigned miRNA include Invitrogen’s StealthTM Select technology (Carlsbad, CA), Ambion® (Austin, TX), and Qiagen® (Valencia, CA). An inhibitory nucleic acid that can be applied in the compositions and methods of the present invention may be any nucleic acid sequence that has been found by any source to be a validated downregulator of a target gene.

[0133] In some embodiments, the miRNA molecule is at least 75, 80, 85, or 90% homologous, particularly at least 95%, 99%, or 100% similar or identical, or any percentages in between the foregoing (e.g., the invention contemplates 75% and greater, 80% and greater, 85% and greater, and so on, and said ranges are intended to include all whole numbers in between), to at least 10 contiguous nucleotides of any of the nucleic acid sequences encoding a full-length protein.

[0134] The miRNA may also comprise an alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material, such as to the end(s) of the 19 to 25 nucleotide RNA or internally (at one or more nucleotides of the RNA). In certain aspects, the RNA molecule contains a 3'-hydroxyl group. Nucleotides in the RNA molecules of the present invention can also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. The double-stranded oligonucleotide may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages. Additional modifications of siRNAs (e.g., 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, “universal base” nucleotides, 5-C- methyl nucleotides, one or more phosphorothioate internucleotide linkages, and inverted deoxyabasic residue incorporation) can be found in U.S. Publication 2004 / 0019001 and U.S. Patent 6,673,611 (each of which is incorporated by reference in its entirety). Collectively, all such altered nucleic acids or RNAs described above are referred to as modified miRNAs.

[0135] In a most preferred embodiment, the miRNA hairpins comprised in the multiplexed miRNA expression construct of the present invention are constructed according to WO201 9186274, in respect of the miRNA architecture and design described therein, which is incorporated by reference herein in its entirety. The skilled person understands how to apply the principles of miRNA design in the art and this reference to achieve optimal results with the multiplexed miRNA expression constructs of the present invention.

[0136] Vectors for Cloning, Gene Transfer and Expression

[0137] Within certain aspects expression vectors are employed to express a nucleic acid of interest, such as a nucleic acid that inhibits the expression of a particular gene. Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers / promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize RNA stability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.

[0138] Regulatory Elements

[0139] Throughout this application, the term “expression construct” or “expression vector” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest i.e., as is the case with RNA molecules of the embodiments.

[0140] In certain embodiments, the nucleic acid encoding a gene product is under transcriptional control of a promoter. A“promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

[0141] The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for eukaryotic RNA polymerase (Pol) I, II or III. Much of the thinking about how promoters are organized derives from analyses of several viral Pol II promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

[0142] At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

[0143] Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

[0144] In some embodiments, the promoter comprises an Elongation Factor 1 short (EF1 s) promoter. In other embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.

[0145] By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product. Tables 3 and 4 list several regulatory elements that may be employed, in the context of the present invention, to regulate the expression of the gene of interest. This list is not intended to be exhaustive of all the possible elements involved in the promotion of gene expression but, merely, to be exemplary thereof. In some aspects, a promoter for use according to the instant embodiments is a non-tissue specific promoter, such as a constitutive promoter.

[0146] Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.

[0147] The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.

[0148] Below is a list of viral promoters, cellular promoters / enhancers and inducible promoters / enhancers that can be used in combination with the nucleic acid encoding a gene or miRNA of interest in an expression construct (Table 3 and Table 4). Additionally, any promoter / enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene or miRNA of interest. Truncated promoters may also be used to drive expression. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

[0149]

[0150] In the context of the present disclosure, any such promoter may be used in combination with the intron for miRNA expression, which may be endogenous or introduced by recombinant modification, e.g. it may be an EF1 a intron in combination with an EF1 a promoter or non-EF1 a promoter. In some highly preferred embodiments, the EF1 a promoter is used. Where any cDNA insert is employed, one will typically include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals. In some aspects, however, a polyadenylation signal sequence is not included in a vector of the embodiments. For example, incorporation of such a signal sequence in lentivira I vectors (before a 3’ LTR) can reduce resulting lentiviral titers. A spacer sequence may be included in the nucleic acid construct. The presence of a spacer appears to enhance knockdown efficiency of miRNA (Stegmeier et al., 2005). Spacers may be any nucleotide sequence. In some aspects, the spacer is GFP.

[0151] Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

[0152] Selectable Markers

[0153] In certain embodiments of the invention, the cells contain nucleic acid constructs of the present invention, a cell may be identified in vitro, ex vivo or in vivo by including a marker in the expression construct. Such markers would confer an identifiable change to the cell permitting easy identification of cells containingthe expression construct. Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed. Immunologic markers also can be employed. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art. In an embodiment, the selectable marker is RQR8, tEGFR or tCD20.

[0154] Delivery of nucleic acid molecules and expression vectors

[0155] In certain aspects, vectors for delivery of nucleic acids of the embodiments could be constructed to express these factors in cells. In a particular aspect, the following systems and methods may be used in delivery of nucleic acids to desired cell types.

[0156] Homologous recombination

[0157] In certain aspects of the embodiments, the vectors encoding nucleic acid molecules of the embodiments may be introduced into cells in a specific manner, for example, via homologous recombination. Current approaches to express genes in stem cells have involved the use of viral vectors (e.g., lentiviral vectors) or transgenes that integrate randomly in the genome. These approaches have not been successful due in part because the randomly integrated vectors can activate or suppress endogenous gene expression, and / or the silencing of transgene expression. The problems associated with random integration could be partially overcome by homologous recombination to a specific locus in the target genome. Homologous recombination (HR), also known as general recombination, is a type of genetic recombination used in all forms of life in which nucleotide sequences are exchanged between two similar or identical strands of DNA. The technique has been the standard method for genome engineering in mammalian cells since the mid-1980s. The process involves several steps of physical breaking and the eventual rejoining of DNA. This process is most widely used in nature to repair potentially lethal double-strand breaks in DNA. In addition, homologous recombination produces new combinations of DNA sequences during meiosis, the process by which eukaryotes make germ cells like sperm and ova. These new combinations of DNA represent genetic variation in offspring which allow populations to evolutionarily adapt to changing environmental conditions over time. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of bacteria and viruses. Homologous recombination is also used as a technique in molecular biology for introducing genetic changes into target organisms.

[0158] Homologous recombination can be used as targeted genome modification. The efficiency of standard HR in mammalian cells is only 10-6to 10-9of cells treated (Capecchi, 1990). The use of meganucleases, or homing endonucleases, such as l-Scel have been used to increase the efficiency of HR. Both natural meganucleases as well as engineered meganucleases with modified targeting specificities have been utilized to increase HR efficiency (Pingoud and Silva, 2007; Chevalier et al., 2002). Another path toward increasing the efficiency of HR has been to engineer chimeric endonucleases with programmable DNA specificity domains (Silva et al., 2011 ). Zinc-finger nucleases (ZFN) are one example of such a chimeric molecule in which Zinc-finger DNA binding domains are fused with the catalytic domain of a Type IIS restriction endonuclease such as Fokl (as reviewed in Durai et al., 2005; PCT / US2004 / 030606). Another class of such specificity molecules includes Transcription Activator Like Effector (TALE) DNA binding domains fused to the catalytic domain of a Type IIS restriction endonuclease such as Fokl (Miller etal., 2011 : PCT / IB2010 / 000154).

[0159] Nucleic acid delivery systems

[0160] One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference). Vectors include but are not limited to, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV etc), lentiviral vectors (e.g., derived from HIV-1 , HIV-2, SIV, BIV, FIV etc.), adenoviral (Ad) vectors including replication competent, replication deficient and gutless forms thereof, adeno- associated viral (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors.

[0161] Episomal Vectors

[0162] The use of plasmid- or liposome-based extra-chromosomal ( / .e., episomal) vectors may be also provided in certain aspects of the invention, for example, for reprogramming of somatic cells. Such episomal vectors may include, e.g., oriP-based vectors, and / or vectors encoding a derivative of EBV-protein EBNA-1. These vectors may permit large fragments of DNA to be introduced to a cell and maintained extra-chromosomally, replicated once per cell cycle, partitioned to daughter cells efficiently, and elicit substantially no immune response.

[0163] In particular, EBNA-1 , the only viral protein required for the replication of the oriP-based expression vector, does not elicit a cellular immune response because it has developed an efficient mechanism to bypass the processing required for presentation of its antigens on MHC class I molecules (Levitskaya et al., 1997). Further, EBNA-1 can act in trans to enhance expression of the cloned gene, inducing expression of a cloned gene up to 100- fold in some cell lines (Langle-Rouault et al., 1998; Evans et al., 1997). Finally, the manufacture of such oriP-based expression vectors is inexpensive.

[0164] Other extra-chromosomal vectors include other lymphotrophic herpes virus-based vectors. Lymphotrophic herpes virus is a herpes virus that replicates in a lymphoblast (e.g., a human B lymphoblast) and becomes a plasmid for a part of its natural life-cycle. Herpes simplex virus (HSV) is not a "lymphotrophic" herpes virus. Exemplary lymphotrophic herpes viruses include, but are not limited to EBV, Kaposi's sarcoma herpes virus (KSHV); Herpes virus saimiri (HS) and Marek's disease virus (MDV). Also other sources of episome-based vectors are contemplated, such as yeast ARS, adenovirus, SV40, or BPV.

[0165] One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (see, for example, Maniatis etal., 1988 and Ausubel etal., 1994, both incorporated herein by reference).

[0166] Vectors can also comprise other components or functionalities that further modulate gene delivery and / or gene expression, or that otherwise provide beneficial properties to the targeted cells. Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide.

[0167] Such components also might include markers, such as detectable and / or selection markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components orfunctionalities mediating binding and uptake), or vectors can be modified to provide such functionalities. A large variety of such vectors are known in the art and are generally available. When a vector is maintained in a host cell, the vector can either be stably replicated by the cells during mitosis as an autonomous structure, incorporated within the genome of the host cell, or maintained in the host cell's nucleus or cytoplasm. Transposon-based system

[0168] According to a particular embodiment the introduction of nucleic acids may use a transposon - transposase system. The used transposon - transposase system could be the well-known Sleeping Beauty, the Frog Prince transposon - transposase system (for the description of the latter see e.g., EP1507865), or the TTAA-specific transposon piggyback system.

[0169] Transposons are sequences of DNA that can move around to different positions within the genome of a single cell, a process called transposition. In the process, they can cause mutations and change the amount of DNA in the genome. Transposons were also once called jumping genes, and are examples of mobile genetic elements.

[0170] There are a variety of mobile genetic elements, and they can be grouped based on their mechanism of transposition. Class I mobile genetic elements, or retrotransposons, copy themselves by first being transcribed to RNA, then reverse transcribed back to DNA by reverse transcriptase, and then being inserted at another position in the genome. Class II mobile genetic elements move directly from one position to another using a transposase to "cut and paste" them within the genome.

[0171] Viral Vectors

[0172] In generating recombinant viral vectors, non-essential genes are typically replaced with a gene or coding sequence for a heterologous (or non-native) protein or nucleic acid. Viral vectors are a kind of expression construct that utilizes viral sequences to introduce nucleic acid and possibly proteins into a cell. The ability of certain viruses to infect cells or enter cells via pH-dependent or pH-independent mechanisms, to integrate their genetic cargo into a host cell genome and to express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of virus vectors that may be used to deliver a nucleic acid of certain aspects of the present invention are described below.

[0173] Retroviruses have promise as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell-lines (Miller, 1992).

[0174] In order to construct a retroviral vector, a nucleic acid is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann eta , 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into a special cell line (e.g., by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid ( / .e., the vector genome) to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Depending on the tropism of the envelope protein used to cover the vector particles surface, retroviral vectors are able to l ' l infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind etal., 1975).

[0175] Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pot, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; Giry-Laterriere et al., 2011 ; U.S. Patents 6,013,516 and 5,994,136).

[0176] Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with one or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Patent 5,994,136, incorporated herein by reference.

[0177] Nucleic acid Delivery

[0178] Introduction of a nucleic acid, such as DNA or RNA, into cells to be programmed with the current invention may use any suitable methods for nucleic acid delivery for transformation of a cell, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection (Wilson etal., 1989, Nabel etal, 1989), by injection (U.S. Patent Nos. 5,994,624, 5,981 ,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference; Tur-Kaspa etal., 1986; Potter etal., 1984); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe etal., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer etal., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley etal., 1979; Nicolau etal., 1987; Wong etal., 1980; Kaneda etal., 1989; Kato etal., 1991) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988); by microprojectile bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Patent Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler etal., 1990; U.S. Patent Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium-med ated transformation (U.S. Patent Nos. 5,591 ,616 and 5,563,055, each incorporated herein by reference); by desiccation / inhibition-mediated DNA uptake (Potrykus etal., 1985), and any combination of such methods. Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.

[0179] Liposome Mediated Transfection

[0180] In a certain embodiment of the invention, a nucleic acid may be entrapped in a lipid complex such as, for example, a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991 ). Also contemplated is a nucleic acid complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen). The amount of liposomes used may vary upon the nature of the liposome as well as the cell used, for example, about 5 to about 20 pg vector DNA per 1 to 10 millions of cells may be contemplated.

[0181] Liposome-mediated nucleic acid delivery and expression of foreign DNA / n vitro has been very successful (Nicolau and Sene, 1982; Fraley etal., 1979; Nicolau etal., 1987). The feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong etal., 1980).

[0182] In certain embodiments of the invention, a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda etal., 1989). In other embodiments, a liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1 ) (Kato etal., 1991 ). In yet further embodiments, a liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In other embodiments, a delivery vehicle may comprise a ligand and a liposome.

[0183] Electroporation

[0184] In certain embodiments of the present invention, a nucleic acid is introduced into an organelle, a cell, a tissue or an organism via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. Recipient cells can be made more susceptible to transformation by mechanical wounding. Also the amount of vectors used may vary upon the nature of the cells used, for example, about 5 to about 20 pg vector DNA per 1 to 10 million of cells may be contemplated.

[0185] Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter etal., 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa etal., 1986) in this manner.

[0186] Calcium Phosphate

[0187] In other embodiments of the present invention, a nucleic acid is introduced to the cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique. Also in this manner, mouse L(A9), mouse C127, CHO, CV-1 , BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe etal., 1990).

[0188] DEAE-Dextran

[0189] In another embodiment, a nucleic acid is delivered into a cell using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985). Cell culturing

[0190] Generally, cells of the present invention are cultured in a culture medium, which is a nutrient-rich buffered solution capable of sustaining cell growth.

[0191] Culture media suitable for isolating, expanding and differentiating stem cells according to the method described herein include but not limited to high glucose Dulbecco's Modified Eagle's Medium (DMEM), DMEM / F-12, Liebovitz L-15, RPMI 1640, Iscove's modified Dubelcco's media (IMDM), and Opti-MEM SFM (Invitrogen Inc.). Chemically Defined Medium comprises a minimum essential medium such as Iscove's Modified Dulbecco's Medium (IMDM) (Gibco), supplemented with human serum albumin, human Ex Cyte lipoprotein, transferrin, insulin, vitamins, essential and non-essential amino acids, sodium pyruvate, glutamine and a mitogen is also suitable. As used herein, a mitogen refers to an agent that stimulates cell division of a cell. An agent can be a chemical, usually some form of a protein that encourages a cell to commence cell division, triggering mitosis. In one embodiment, serum free media such as those described in U.S. Ser. No. 08 / 464,599 and WO96 / 39487, and the "complete media" as described in U.S. Pat. No. 5,486,359 are contemplated for use with the method described herein. In some embodiments, the culture medium is supplemented with 10% Fetal Bovine Serum (FBS), human autologous serum, human AB serum or platelet rich plasma supplemented with heparin (2U / ml). Cell cultures may be maintained in a CO2atmosphere, e.g., 5% to 12%, to maintain pH of the culture fluid, incubated at 37°C in a humid atmosphere and passaged to maintain a confluence below 85%.

[0192] Numbered paragraphs

[0193] The disclosure can further be understood by reference to the embodiments set forth in the following numbered paragraphs:

[0194] 1 . A microRNA (miRNA) expression construct comprising a promoter operably linked to an expressed transcript, wherein the promoter comprises a promoter element and an intronic element, wherein the intronic element comprises one or more miRNA hairpins.

[0195] 2. The miRNA expression construct of paragraph 1 , wherein the intronic element is an elongation factor 1-alpha (EF1 a) intron, derivative or fragment thereof.

[0196] 3. The miRNA expression construct of any preceding paragraph, wherein the intronic element comprises the sequence set forth in SEQ ID NO: 3 or a sequence having at least 80% identity thereto.

[0197] 4. The miRNA expression construct of any preceding paragraph, wherein the promoter is an EF1 a promoter, a derivative of a EF1 a promoter or a EF1 short promoter, optionally wherein the EF1 a promoter comprises the sequence set forth in SEQ ID NO: 1 or a sequence having at least 80% identity thereto.

[0198] 5. The miRNA expression construct of any one of paragraphs 1 to 3, wherein the promoter is a eukaryotic promoter, such as a Pol II or Pol III promoter, an inducible promoter, tissue-specific promoter, cell lineage-specific promoter, a synthetic promoter, and / or a promoter listed in Table 3 or a UBI promoter.

[0199] 6. The miRNA expression construct of any preceding paragraph, wherein the promoter is a synthetic promoter further comprising an elongation factor 1-alpha (EF1 a) intron, derivative or fragment thereof, optionally comprising SEQ ID NO: 3 or a sequence having at least 80% identity thereto.

[0200] 7. The miRNA expression construct of any preceding paragraph, wherein the promoter comprises the sequence setforth in SEQ ID NO: 1 or a sequence having at least 80% identity thereto.

[0201] 8. The miRNA expression construct of any preceding paragraph, wherein the intronic sequence comprises at least four, or at least six miRNA hairpins

[0202] 9. The miRNA expression construct of any preceding paragraph, wherein the miRNA hairpins target at least two, at least three, at least four, at least five or at least six different transcripts or genes.

[0203] 10. The miRNA expression construct of any preceding paragraph, wherein each of the miRNA hairpins functionally modulates a target gene, optionally wherein each of the miRNA hairpins provides a level of functional modulation of its target gene that is the same or equivalent to the miRNA expression construct that only comprises that miRNA hairpin.

[0204] 11 . The miRNA expression construct of any preceding paragraph, wherein the intronic element comprises miRNA hairpins targeting either: a) one or more of Cd3z, B2M, CD7, CD5 and / or CIITA; or b) one or more of Cd3z, CD52, B2M, CD7, CD5 and / or CIITA.

[0205] 12. The miRNA expression construct of any preceding paragraph, wherein the intronic element comprises a first and second miRNA hairpin targeting Cd3z, a miRNA hairpin targeting B2M and a miRNA hairpin targeting CD7, optionally wherein the intronic sequence further comprises a miRNA hairpin targeting CD5 and a miRNA hairpin targeting CIITA.

[0206] 13. The miRNA expression construct of any preceding paragraph, wherein the intronic element comprises a miRNA hairpin targeting CD52, a miRNA hairpin targeting B2M and a miRNA hairpin targeting CD3z.

[0207] 14. The miRNA expression construct of any preceding paragraph, wherein a miRNA hairpin targeting CD3z comprises a sequence having 80% sequence identity to SEQ ID NO: 4, or a fragment thereof; and / or a miRNA hairpin targeting CD3z comprises a sequence having 80% sequence identity to SEQ ID NO: 5, or a fragment thereof; and / or a hairpin targeting CD52 comprises a sequence having 80% sequence identity to SEQ ID NO 12, or a fragment thereof; and / or a hairpin targeting B2M comprises a sequence having 80% sequence identity to SEQ ID NO: 6, or a fragment thereof; and / or a hairpin targeting CD7 comprises a sequence having 80% sequence identity to SEQ ID NO: 7, or a fragment thereof; and / or a hairpin targeting CD5 comprises a sequence having 80% sequence identity to SEQ ID NO: 8, or a fragment thereof; and / or a hairpin targeting CIITA comprises a sequence having 80% sequence identity to SEQ ID NO: 9, or a fragment thereof.

[0208] 15. The miRNA expression construct of any preceding paragraph, wherein the construct comprises at least two different miRNA hairpins which target different regions of the same transcript, and / or which target different transcripts or splice variants of the same gene.

[0209] 16. The miRNA expression construct of any preceding paragraph, wherein the expressed transcript comprises a sequence encoding a moiety for re-directing immune effector cell function.

[0210] 17. The miRNA expression construct of any preceding paragraph, wherein the miRNA expression construct comprises a sequence encoding an engineered T cell receptor, optionally a chimeric antigen receptor.

[0211] 18. The miRNA expression construct of any preceding paragraph, wherein the chimeric antigen receptor specifically binds to CD7, optionally wherein the chimeric antigen receptor comprises an anti-CD7 VHH.

[0212] 19. The miRNA expression construct of any preceding paragraph, wherein the chimeric antigen receptor specifically binds to CD19, optionally wherein the chimeric antigen receptor comprises an anti-CD19 VHH or scFv, preferably an anti-CD19 scFv.

[0213] 20. The miRNA expression construct of paragraph 17, wherein the chimeric antigen receptor is a bispecific chimeric antigen receptor or comprised of tandemly expressed chimeric antigen receptors for dual targeting of antigens on tumor tissues.

[0214] 21. The miRNA expression construct of any preceding paragraph, wherein the construct further comprises a selection gene, optionally LNGFR, truncated endothelial growth factor receptor (tEGFR), tCD19, tCD20, tCD34 or a derivative thereof.

[0215] 22. The miRNA expression construct of any preceding paragraph, wherein the construct further comprises a suicide gene, optionally herpes simplex virus thymidine kinase (HSV-tk), inducible caspase 9 (iCasp9), truncated endothelial growth factor receptor (tEGFR), RQR8, dihydrofolate reductase (DHFR), tCD20 and thymidylate synthase (TYMS).

[0216] 23. The miRNA expression construct of any preceding paragraph, wherein the construct further comprises an internal ribosome entry site (IRES).

[0217] 24. The miRNA expression construct of any preceding paragraph, wherein the construct further comprises a peptide cleavage site, optionally a 2A peptide, optionally 2A, P2A, T2A, E2A, F2A, BmCPV 2A, and BmIFV 2A. 25. The miRNA expression construct of any preceding paragraph, wherein the construct achieves knockdown or functional silencing of at least one, two, three, four, five or six genes.

[0218] 26. A nucleic acid comprising the miRNA expression construct of any preceding paragraph.

[0219] 27. A plasmid comprising the miRNA expression construct or nucleic acid of any preceding paragraph.

[0220] 28. A vector comprising the miRNA expression construct, nucleic acid or plasmid of any preceding paragraph.

[0221] 29. The vector of paragraph 28, wherein the vector is an expression vector, preferably an adenovirus, an adeno-associated virus, a retrovirus or a lentivirus vector.

[0222] 30. The vector of paragraph 28 or 29, further comprising at least one drug resistance marker.

[0223] 31 . A host cell comprising the miRNA expression construct, nucleic acid, plasmid or vector of any preceding paragraph.

[0224] 32. The host cell of paragraph 31 , wherein the host cell is a eukaryotic cell, preferably a mammalian cell, preferably an immune effector cell.

[0225] 33. The host cell of paragraph 32, wherein the immune effector cell is selected from the group comprising: alpha-beta T cells, gamma-delta T-cells, tumour infiltrating lymphocytes (TILS), TCR-engineered T cells, CART cells, NK cells, NK / T cells, T regulatory cells, monocytes and macrophages.

[0226] 34. The host cell of paragraph 33, wherein the host cell is a T-cell or a CAR T-cell.

[0227] 35. A composition comprising the miRNA expression construct, nucleic acid, plasmid, vector or host cell of any preceding paragraph.

[0228] 36. The composition of paragraph 35, wherein the composition further comprises one or more pharmaceutically acceptable adjuvants, diluents, excipients or salts.

[0229] 37. A method for reducing expression of a gene in a cell comprising expressing the miRNA expression construct, nucleic acid, plasmid or vector of any one of paragraphs 1 to 30 in the cell.

[0230] 38. The method of paragraph 37, wherein expressing the miRNA expression construct, nucleic acid, plasmid or vector in the cell comprises transfecting the cell with the miRNA expression construct, nucleic acid, plasmid or vector. 39. The method of paragraph 37 or 38, wherein expressing the miRNA expression construct, nucleic acid, plasmid or vector in the cell comprises introducing the miRNA expression construct, nucleic acid, plasmid orvector into the cell via electroporation.

[0231] 40. The method of any one of paragraphs 37 to 39, wherein expressing the miRNA expression construct, nucleic acid, plasmid orvector in the cell comprises transfecting the cell with a transposon comprising the miRNA expression construct, nucleic acid, plasmid or vector, optionally wherein the transposon is sleeping beauty, piggyBAC or To 12.

[0232] 41 . The method of any one of paragraphs 37 to 40, wherein the cell is a eukaryotic cell, preferably a human cell, preferably an immune effector cell.

[0233] 42. The method of any one of paragraphs 37 to 41 , wherein the method is an in vivo, in vitro or ex vivo method.

[0234] 43. The method of any one of paragraphs 37 to 42, further comprising transplanting the cell into an organism.

[0235] 44. The method of any one of paragraphs 37 to 43, wherein the cell is comprised in an organism.

[0236] 45. A method for preparing an engineered cell, optionally an immune effector cell, from a patient or healthy donor comprising:

[0237] (a) collecting a cell, e.g. from the patient ;

[0238] (b) gene modifying the cell to generate an engineered cell: i) with a chimeric antigen receptor or a T cell receptor; and ii) with the multiplexed miRNA from the same expression construct, nucleic acid, plasmid orvector of any one of paragraphs 1 to 30.

[0239] 46. An engineered effector cell obtainable by the method of paragraph 45.

[0240] 47. The method of paragraph 45, wherein the miRNA expression construct, nucleic acid, plasmid or vector is inserted into the cell by a TALE nuclease, megaTAL, Zinc Finger Nuclease, or CRISPR, optionally CRISPR / Cas9.

[0241] 48. The method of paragraph 45 or 46, wherein the miRNA expression construct, nucleic acid, plasmid or vector is expressed in the cell from an expression vector, optionally an adenovirus, an adeno-associated virus, a retrovirus or a lentivirus vector.

[0242] 49. The method of any one of paragraphs 45 to 47, wherein the miRNA expression construct, nucleic acid, plasmid or vector is expressed in the cell from a transposon, optionally wherein the transposon is sleeping beauty, piggyBAC orTol2.

[0243] 50. The miRNA expression construct, nucleic acid, plasmid, vector, composition or host cell of any one of paragraphs 1 to 36 or 46, for use in therapy. 51 . The miRNA expression construct, nucleic acid, plasmid, vector, composition, or host cell of any one of paragraphs 1 to 36 or 46, for use in a method of treating cancer, an infectious disease, an auto-immune disease or an inherited disorder.

[0244] 52. A method of treating cancer, an infectious disease, an auto-immune disease or an inherited disorder, comprising administering the miRNA expression construct, nucleic acid, plasmid, vector, composition, or host cell of any one of paragraphs 1 to 36 or 46.

[0245] 53. The miRNA expression construct, nucleic acid, plasmid, vector, composition, or host cell of any one of paragraphs 1 to 36 or 46, for use in a method of manufacturing a medicament.

[0246] 54. The miRNA expression construct, nucleic acid, plasmid, vector, composition, or host cell of any one of paragraphs 1 to 36 or 46, for use in a method of manufacturing a medicament for the treatment of cancer, an infectious disease, an auto-immune disease or an inherited disorder.

[0247] 55. A method of making a miRNA expression construct, comprising: a) providing an intronic element sequence; b) modifying the intronic element sequence by inserting one or more miRNA hairpins into the intronic element sequence; c) incorporating the intronic element sequence and a promoter element into a promoter; and d) providing a nucleic acid comprising the promoter operably linked to an expressed transcript.

[0248] Examples

[0249] Methodology

[0250] Molecular cloning of plasmids and lentiviral vectors.

[0251] The modified elongation factor 1 alpha promoter, named hmEFI a and our proprietary microRNAs were synthesized by ThermoFisher Scientific (Table 1 ). The microRNAs (Hairpins = HPs) were subcloned, using the restriction enzymes BamHI / Xbal, into the intron of our modified EF1 a promoter. The plasmids generated (pENTR-L4-hmEF1 a-L1 R) were subcloned with either 3HPs, 4 HPs or 6HPs. The final lentivector plasmidswere generated by an LR Clonase II (Invitrogen, Carlsbad, CA)-mediated recombination of the pENTR plasmid containing the hmEFI a promoter (pENTR-L4-hmEF1 a-L1 R), a pENTR plasmid containing the GOI (Gene Of Interest), in our case CD7-CAR (pENTR-L1-CD7- CAR-L2) or CD19-CAR (pENTR-L1-CD19-CAR-L2), and a pCLX-R4-DEST-R2 (Figure 1 ) or a pCWX-R4-DEST-R2-PGK-mCherry lentivector destination cassette (Figure 4).

[0252] Lentiviral vector production

[0253] Lentiviral vectors carrying the RQR8 reporter gene were produced by transfecting HEK293T cells with transfer plasmids carrying the gene silencing construct, as well as lentiviral packaging (PAX2) and envelope (VSVg) plasmids. The cell culture medium was replenished after 4-6 hours and subsequently harvested at 24 and 48 hours for viral particle collection. After the culture medium was collected, it was filtered to remove cellular debris and concentrated using PEG-lt Virus Precipitation Solution (System Biosciences), according to the manufacturer’s instructions. Final aliquots of concentrated lentiviral vectors were stored at -80°C. Functional viral vector titers were assessed by transducing HT1080 cells over a range of dilutions and measuring the percentage of cells expressing RQR8 reporter gene.

[0254] Cells and cell lines

[0255] The expression and silencing of all gene constructs was assessed in primary T-cells, which were prepared from anonymized buffy coat blood units procured from the Blood Transfusion Centre of the University Hospital of Geneva, Switzerland. The peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll separation, after which T- cells were separated using EasySep™ Human T Cell Isolation Kit (StemCell) and cryopreserved in aliquots in liquid nitrogen.

[0256] Gene delivery and CAR T-cell manufacturing

[0257] Cryopreserved T-cells were thawed, cultured in T-cell medium (Xvivo, 5% Human Serum, 1 % penicillin / streptomycin) complemented with IL-7 and IL-15 (10ng / mL each), and activated using CD3 / CD28 Dynabeads, as per manufacturer’s instructions. Activated T- cells were transduced 24 hours later with lentiviral vectors carrying the miRNA gene silencing constructs (for mirGE screening) and miRNA gene silencing and CAR expression constructs (for CAR T-cell production). Transductions were performed in high density volumes (1 million cells per mL for CAR T-cell production in GREX plates. For CAR T-cell production, medium was added to top up the well 24h after transduction and 8 days after transduction cells were re-seeded in a new GREX plate (in a 24-well GREX to re-seed between 0.5-1 million cells and in a 6-well GREX to re-seed between 5-15 million cells). When cells were re-seeded in a 24-well GREX, half of the medium was exchanged on D12 for fresh medium. For CAR T cell production and on day 15 after transduction, TCR depletion (EasySep™ Human TCR Alpha / Beta Depletion Kit, StemCell) was performed and cells were frozen and stored in liquid nitrogen. During this production process, silencing, transduction and memory, exhaustion and activation markers were assessed by flow cytometry at day 5 and 17 after transduction and post-thaw.

[0258] Flow cytometry and assessment of gene silencing

[0259] Flow cytometry was performed at 5 and 17 days post-transduction, and at post-thaw. Cells were harvested, washed, resuspended in Staining buffer (EasySep™ Human TCR Alpha / Beta Depletion Kit (Miltenyi Biotec), 5% BSA), and stained for 10-30 min with the appropriate antibodies for assessment of cell surface expression. Following staining, cells were washed with Staining buffer, resuspended in Staining buffer, and cell surface expression assessed via flow cytometry. Captured data were exported to FlowJo for analysis. To calculate the level of gene silencing, changes in both the percentage of cells positive for the target and median florescence intensity (MFI) were assessed. Normalization includes expression levels within samples (modified vs unmodified cells), after which expression levels relative to the control-transduced cells were calculated. Cytotoxicity assays

[0260] Cell lines MOLT-4 (ATCC CRL-1582), SUP-T1 (ATCC CRL-192) and Jeko-1 stably expressing the fluorescent protein GFP (ATCC CRL-3000) were purchased from ATCC. Tumor cells stained with CellTrace Violet (Thermo Fisher Scientific) or stably expressing GFP were mixed with untransduced or CAR T cells at effector to target ratios 3:1 , 1 :1 or 1 :3. For recursive killing assays, the number of tumor cells and T-cells were assessed every 3-4 days by flow cytometry. At each timepoint and upon tumor cell clearance, the same number as initially plated of new tumor cells was added to T cells.

[0261] Results and Discussion

[0262] We developed novel gene constructs expressing intronic miRNAs by exploiting the natural architecture of the human EF1-alpha promoter. In proof of principle experiments, we multiplex engineered CAR T-cells co-expressing either four or six intronic miRNAs, which were characterized immunophenotypically and functionally via recursive cytoxicity of tumor cells.

[0263] Multiplex gene constructs were initially designed using a second-generation anti-CD7 CAR incorporating a single-domain antibody (VHH) as the antigen-binding region (as detailed in GB2402745.0, incorporated by reference herein), and a second-generation anti-CD19 CAR incorporating a single-chain variable fragment (scFv) as the binder domain. . The human EF1-alpha promoter, driving CAR expression in both constructs, was truncated to preserve relevant flanking regions of the existing first intron (Table 5). Optimized miRNAs for silencing gene expression of appropriate targets, namely CD3<^, 02M, OITA, CD7 and CD5, were subsequently cloned to replace the truncated region (Figure 1 , Table 5). We created two versions of anti-CD7 CAR construct for testing, the first carrying four intronic miRNAs and the second carrying six. Additionally, one version of anti-CD19 CAR construct carrying three intronic miRNAs was created. All constructions also co-expressed RQR8 as a reporter gene, and a second reporter gene mCherry expressed through a second constitutive promoter, hPGK, located downstream the modified EF1-alpha promoter. The gene constructs were packaged into lentiviral vectors for gene modification of primary T-cells.

[0264] CD7 CAR T-cells, co-expressing intronic miRNAs, were produced via lentiviral vector transduction, cell culture expanded for 15 days and purified via depletion of remaining TCRa / p positive cells. Phenotypically, CD7 CAR and RQR8 (CD34) expressed in >90% of modified T-cells (Figure 2A-B), confirming efficient gene transfer. Silencing of TCR, HLA-I and CD7 was shown to be very efficient in both the four and six intronic miRNAs modified T-cells. In addition, the six intronic miRNAs modified cells showed efficient silencing on the two additional targets, namely HLA-II and CD5.

[0265] To verify the functional activity of the CD7 CAR T-cells, we performed long-term cytoxicity assays against two different CD7-expressing cell lines. These long-term assays were based on recursive stimulation of the CAR T-cells by the different tumor cell lines. Both the four and six intronic miRNA expressing CAR T-cells were shown to be efficient at eliminating MOLT-4 and SUP-T1 leukemic cells, at least two times in a row (Figure 3). CD19 CAR T-cells, co-expressing intronic miRNAs, were produced via lentiviral vector transduction and subsequently expanded in culture for five days prior to flow cytometry analysis. Phenotypic characterization identified CD19 CAR-positive cells via direct detection of RQR8 co-expression using an anti-CD34 antibody. With approximately 59.8% of modified T cells (Figure 5A-B), these results demonstrate an efficient gene transfer. Moreover, the proportion of mCherry-positive cells (average 62.5%) confirms robust expression from the downstream promoter and further validates the effectiveness of the gene transfer. Furthermore, silencing of CD52, HLA-I and TCR was effectively achieved in T-cells modified with three intronic miRNAs.

[0266] To verify the functional activity of the CD19 CAR T-cells, short-term cytoxicity assays against CD19-expressing cell lines at different ratios (3:1 , 1 :1 and 1 :3) were performed. These short-term assays were based on 2- or 3-days stimulation of the CAR T-cells by the tumor cell lines. CD19 CAR T-cell populations, expressing three intronic miRNAs were shown to be efficient at eliminating Jeko1-GFP cells (Figure 6) at E:T ratio of 3:1 and 1 :1 and able to control the tumor growth at an E:T ratio of 1 :3.

[0267] Taken together, these data not only demonstrate that the newly constructed intronic miRNAs could be efficiently expressed and used to functionally modulate (e.g. silence) multiple different target genes from a modified EF1-alpha promoter without compromising CAR expression (for both CD7 and CD19 CAR), but also that the modified cells maintained effective functional activity in long-term / stress assays against aggressive tumor cell lines. Surprisingly, functionality, CAR and miRNA expression were obtained even with as many as four or six intronic miRNAs, and multiple different targets were modulated.

[0268] More broadly, although the principle of expressing intronic miRNAs in multiplex- engineered CAR T-cells is demonstrated herein, these optimized gene constructs can be applied in various other contexts where gene or gene-modified cell therapies are developed. The novel EF1-alpha intron , which is very efficiently spliced prior to Drosha processing and destabilization of the protein-expressing modules, indicates that it can likely be adapted further to express even more miRNAs (up to 10). The intron can thus also be relevant for expression of multiplexed miRNAs of varying architectures (naturally derived or synthetic), and not limited to the mirGE architecture reported here (Myburgh et al., 2014)

[0269] Tables

[0270] Table 5. DNA sequences used for gene construction of intronic miRNA

[0271] References

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Claims

Claims1 . A microRNA (miRNA) expression construct comprising a promoter operably linked to an expressed transcript, wherein the promoter comprises a promoter element and an intronic element, and wherein the intronic element comprises one or more miRNA hairpins.

2. The miRNA expression construct of claim 1 , wherein the intronic element is an elongation factor 1-alpha (EF1 a) intron, derivative or fragment thereof.

3. The miRNA expression construct of any preceding claim, wherein the intronic element comprises the sequence set forth in SEQ ID NO: 3 or a sequence having at least 80% identity thereto.

4. The miRNA expression construct of any preceding claim, wherein the promoter is an EF1 a promoter, a derivative of an EF1 a promoter or an EF1 short promoter, optionally wherein the EF1 a promoter comprises the sequence set forth in SEQ ID NO: 1 or a sequence having 80% identity thereto.

5. The miRNA expression construct of any one of claims 1 to 3, wherein the promoter is an eukaryotic promoter, such as a Pol II or Pol III promoter, an inducible promoter, tissue-specific promoter, cell lineage-specific promoter, a synthetic promoter, and / or a promoter listed in Table 3 or a UBI promoter.

6. The miRNA expression construct of any preceding claim, wherein the promoter is a synthetic promoter further comprising an elongation factor 1-alpha (EF1 a) intron, derivative or fragment thereof, optionally comprising SEQ ID NO: 3 or a sequence having at least 80% identity thereto.

7. The miRNA expression construct of any preceding claim, wherein the promoter comprises the sequence set forth in SEQ ID NO: 1 or a sequence having at least 80% identity thereto.

8. The miRNA expression construct of any preceding claim, wherein the intronic sequence comprises at least four, or at least six miRNA hairpins9. The miRNA expression construct of any preceding claim, wherein the miRNA hairpins target at least two, at least three, at least four, at least five or at least six different target genes or transcripts.

10. The miRNA expression construct of any preceding claim, wherein each of the miRNA hairpins functionally modulates a target gene, optionally wherein, for each of the miRNA hairpins, the miRNA expression construct comprising said miRNA hairpin provides a level of functional modulation of its target gene that is the same or equivalent to a miRNA expression construct that only comprises that miRNA hairpin.

11. The miRNA expression construct of any preceding claim, wherein the intronic element comprises miRNA hairpins targeting one or more ofCD3z, CD52, B2M, CD7, CD5 and / or CIITA.

12. The miRNA expression construct of any preceding claim, wherein the intronic element comprises a first and second miRNA hairpin targeting CD3z, a miRNA hairpin targeting B2M and a miRNA hairpin targeting CD7, optionally wherein the intronic sequence further comprises a miRNA hairpin targeting CD5 and a miRNA hairpin targeting OITA.

13. The miRNA expression construct of any preceding claim, wherein the intronic element comprises a miRNA hairpin targeting CD52, a miRNA hairpin targeting B2M and a miRNA hairpin targeting CD3z.

14. The miRNA expression construct of any preceding claim, wherein a miRNA hairpin targeting CD3z comprises a sequence having 80% sequence identity to SEQ ID NO: 4, or a fragment thereof; and / or a miRNA hairpin targeting CD3z comprises a sequence having 80% sequence identity to SEQ ID NO: 5, or a fragment thereof; and / or a hairpin targeting CD52 comprises a sequence having 80% sequence identity to SED ID NO 12, or a fragment thereof; and / or a hairpin targeting B2M comprises a sequence having 80% sequence identity to SEQ ID NO: 6, or a fragment thereof; and / or a hairpin targeting CD7 comprises a sequence having 80% sequence identity to SEQ ID NO: 7, or a fragment thereof; and / or a hairpin targeting CD5 comprises a sequence having 80% sequence identity to SEQ ID NO: 8, or a fragment thereof; and / or a hairpin targeting CIITA comprises a sequence having 80% sequence identity to SEQ ID NO: 9, or a fragment thereof.

15. The miRNA expression construct of any preceding claim, wherein the expressed transcript comprises a sequence encoding a moiety for re-directing immune effector cell function; and / or the miRNA expression construct comprises a sequence encoding an engineered T cell receptor, optionally a chimeric antigen receptor, optionally wherein the chimeric antigen receptor specifically binds to CD7 and / or CD19, optionally wherein the chimeric antigen receptor comprises an anti-CD7 VHH or an anti-CD19 scFv.

16. The miRNA expression construct of any preceding claim, wherein the chimeric antigen receptor is a bispecific chimeric antigen receptor or comprised of tandemly expressed chimeric antigen receptors for dual targeting of antigens on tumor tissues.

17. The miRNA expression construct of any preceding claim, wherein the construct achieves knockdown or functional silencing of at least one, two, three, four, five or six genes.

18. A nucleic acid, plasmid or vector comprisingthe miRNA expression construct of any preceding claim; optionally wherein the vector is an expression vector, preferably an adenovirus, an adeno-associated virus, a retrovirus or a lentivirus vector.

19. A host cell comprisingthe miRNA expression construct, nucleic acid, plasmid or vector of any preceding claim; optionally wherein the host cell is a eukaryotic cell, preferably a mammalian cell, preferably an immune effector cell; optionally wherein the immune effector cell is selected from the group comprising: alpha-beta T cells, gammadelta T-cells, tumour infiltrating lymphocytes (TILS), TCR-engineered T cells, CART cells, NK cells, NK / T cells, T regulatory cells, monocytes and macrophages.

20. A composition comprising the miRNA expression construct, nucleic acid, plasmid, vector or host cell of any preceding claim; optionally wherein the composition further comprises one or more pharmaceutically acceptable adjuvants, diluents, excipients or salts.21 . A method for reducing expression of a gene in a cell comprising expressing the miRNA expression construct, nucleic acid, plasmid or vector of any one of claims 1 to 18 in the cell.

22. A method for preparing an engineered cell, optionally an immune effector cell, from a patient or healthy donor comprising:(a) collecting a cell, e.g. from the patient;(b) gene modifying the cell to generate an engineered cell: i) with a chimeric antigen receptor or a T cell receptor; and ii) with the multiplexed miRNA from the same expression construct, nucleic acid, plasmid or vector of any one of claims 1 to 18.

23. An engineered cell obtainable by the method of claim 22.

24. The miRNA expression construct, nucleic acid, plasmid, vector, composition or host cell of any one of claims 1 to 19 or 23, for use in therapy.

25. The miRNA expression construct, nucleic acid, plasmid, vector, composition, or host cell of any one of claims 1 to 19 or 23, for use in a method of treating cancer, an infectious disease, an auto-immune disease or an inherited disorder.

26. A method of making a miRNA expression construct, comprising: a) providing an intronic element sequence; b) modifyingthe intronic element sequence by inserting one or more miRNA hairpins into the intronic element sequence; c) incorporatingthe intronic element sequence and a promoter element into a promoter; and d) providing a nucleic acid comprisingthe promoter operably linked to an expressed transcript.46

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